Interactions between Fkh1 monomers stabilize its binding to DNA replication origins.

Eukaryotic DNA replication is initiated from multiple genomic origins, which can be broadly categorized as firing early or late in the S phase. Several factors can influence the temporal usage of origins to determine the timing of their firing. In budding yeast, the Forkhead family proteins Fkh1 and Fkh2 bind to a subset of replication origins and activate them at the beginning of the S phase. In these origins, the Fkh1/2 binding sites are arranged in a strict configuration, suggesting that Forkhead factors must bind the origins in a specific manner. To explore these binding mechanisms in more detail, we mapped the domains of Fkh1 that were required for its role in DNA replication regulation. We found that a short region of Fkh1 near its DNA binding domain was essential for the protein to bind and activate replication origins. Analysis of purified Fkh1 proteins revealed that this region mediates dimerization of Fkh1, suggesting that intramolecular contacts of Fkh1 are required for efficient binding and regulation of DNA replication origins. We also show that the Sld3-Sld7-Cdc45 complex is recruited to Forkhead-regulated origins already in the G1 phase and that Fkh1 is constantly required to keep these factors bound on origins before the onset of the S phase. Together, our results suggest that dimerization-mediated stabilization of DNA binding by Fkh1 is crucial for its ability to activate DNA replication origins.

Transcriptional regulator Taf14 binds DNA and is required for the function of transcription factor TFIID in the absence of histone H2A.Z.

The transcriptional regulator Taf14 is a component of multiple protein complexes involved in transcription initiation and chromatin remodeling in yeast cells. Although Taf14 is not required for cell viability, it becomes essential in conditions where the formation of the transcription preinitiation complex is hampered. The specific role of Taf14 in mediating transcription initiation and preinitiation complex formation is unclear. Here, we explored its role in the general transcription factor IID by mapping Taf14 genetic and proteomic interactions and found that it was needed for the function of the complex if Htz1, the yeast homolog of histone H2A.Z, was absent from chromatin. Dissecting the functional domains of Taf14 revealed that the linker region between the YEATS and ET domains was required for cell viability in the absence of Htz1 protein. We further show that the linker region of Taf14 interacts with DNA. We propose that providing additional DNA binding capacity might be a general role of Taf14 in the recruitment of protein complexes to DNA and chromatin.

MPK12 in stomatal CO2 signaling: function beyond its kinase activity.

Protein phosphorylation is a major molecular switch involved in the regulation of stomatal opening and closure. Previous research defined interaction between MAP kinase 12 and Raf-like kinase HT1 as a required step for stomatal movements caused by changes in CO2 concentration. However, whether MPK12 kinase activity is required for regulation of CO2 -induced stomatal responses warrants in-depth investigation. We apply genetic, biochemical, and structural modeling approaches to examining the noncatalytic role of MPK12 in guard cell CO2 signaling that relies on allosteric inhibition of HT1. We show that CO2 /HCO3 - -enhanced MPK12 interaction with HT1 is independent of its kinase activity. By analyzing gas exchange of plant lines expressing various kinase-dead and constitutively active versions of MPK12 in a plant line where MPK12 is deleted, we confirmed that CO2 -dependent stomatal responses rely on MPK12's ability to bind to HT1, but not its kinase activity. We also demonstrate that purified MPK12 and HT1 proteins form a heterodimer in the presence of CO2 /HCO3 - and present structural modeling that explains the MPK12:HT1 interaction interface. These data add to the model that MPK12 kinase-activity-independent interaction with HT1 functions as a molecular switch by which guard cells sense changes in atmospheric CO2 concentration.

Stalled fork rescue via dormant replication origins in unchallenged S phase promotes proper chromosome segregation and tumor suppression.

Eukaryotic cells license far more origins than are actually used for DNA replication, thereby generating a large number of dormant origins. Accumulating evidence suggests that such origins play a role in chromosome stability and tumor suppression, though the underlying mechanism is largely unknown. Here, we show that a loss of dormant origins results in an increased number of stalled replication forks, even in unchallenged S phase in primary mouse fibroblasts derived from embryos homozygous for the Mcm4(Chaos3) allele. We found that this allele reduces the stability of the MCM2-7 complex, but confers normal helicase activity in vitro. Despite the activation of multiple fork recovery pathways, replication intermediates in these cells persist into M phase, increasing the number of abnormal anaphase cells with lagging chromosomes and/or acentric fragments. These findings suggest that dormant origins constitute a major pathway for stalled fork recovery, contributing to faithful chromosome segregation and tumor suppression.

Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins.

MCM2-7 proteins provide essential helicase functions in eukaryotes at chromosomal DNA replication forks. During the G1 phase of the cell cycle, they remain loaded on DNA but are inactive. We have used recombinant methods to show that the Drosophila MCM2-7 helicase is activated in complex with Cdc45 and the four GINS proteins (CMG complex). Biochemical activities of the MCM AAA+ motor are altered and enhanced through such associations: ATP hydrolysis rates are elevated by two orders of magnitude, helicase activity is robust on circular templates, and affinity for DNA substrates is improved. The GINS proteins contribute to DNA substrate affinity and bind specifically to the MCM4 subunit. All pairwise associations among GINS, MCMs, and Cdc45 were detected, but tight association takes place only in the CMG. The onset of DNA replication and unwinding may thus occur through allosteric changes in MCM2-7 affected by the association of these ancillary factors.

Checkpoint kinase 2 (Chk2) inhibits the activity of the Cdc45/MCM2-7/GINS (CMG) replicative helicase complex.

The replication of eukaryote chromosomes slows down when DNA is damaged and the proteins that work at the fork (the replisome) are known targets for the signaling pathways that mediate such responses critical for accurate genomic inheritance. However, the molecular mechanisms and details of how this response is mediated are poorly understood. In this report we show that the activity of replisome helicase, the Cdc45/MCM2-7/GINS (CMG) complex, can be inhibited by protein phosphorylation. Recombinant Drosophila melanogaster CMG can be stimulated by treatment with phosphatase whereas Chk2 but not Chk1 interferes with the helicase activity in vitro. The targets for Chk2 phosphorylation have been identified and reside in MCM subunits 3 and 4 and in the GINS protein Psf2. Interference requires a combination of modifications and we suggest that the formation of negative charges might create a surface on the helicase to allosterically affect its function. The treatment of developing fly embryos with ionizing radiation leads to hyperphosphorylation of Psf2 subunit in the active helicase complex. Taken together these data suggest that the direct modification of the CMG helicase by Chk2 is an important nexus for response to DNA damage.

A novel role of KEAP1/PGAM5 complex: ROS sensor for inducing mitophagy.

When ROS production exceeds the cellular antioxidant capacity, the cell needs to eliminate the defective mitochondria responsible for excessive ROS production. It has been proposed that the removal of these defective mitochondria involves mitophagy, but the mechanism of this regulation remains unclear. Here, we demonstrate that moderate mitochondrial superoxide and hydrogen peroxide production oxidates KEAP1, thus breaking the interaction between this protein and PGAM5, leading to the inhibition of its proteasomal degradation. Accumulated PGAM5 interferes with the processing of the PINK1 in the mitochondria leading to the accumulation of PINK1 on the outer mitochondrial membrane. In turn, PINK1 promotes Parkin recruitment to mitochondria and sensitizes mitochondria for autophagic removal. We also demonstrate that inhibitors of the KEAP1-PGAM5 protein-protein interaction (including CPUY192018) mimic the effect of mitochondrial ROS and sensitize mitophagy machinery, suggesting that these inhibitors could be used as pharmacological regulators of mitophagy. Together, our results show that KEAP1/PGAM5 complex senses mitochondrially generated superoxide/hydrogen peroxide to induce mitophagy.

Keap1-MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa.

Coordination of DNA replication and cellular redox homeostasis mechanisms is essential for the sustained genome stability due to the sensitivity of replicating DNA to oxidation. However, substantial gaps remain in our knowledge of underlying molecular pathways. In this study, we characterise the interaction of Keap1, a central antioxidant response regulator in Metazoa, with the replicative helicase subunit protein MCM3. Our analysis suggests that structural determinants of the interaction of Keap1 with its critical downstream target - Nrf2 master transactivator of oxidative stress response genes - may have evolved in evolution to mimic the conserved helix-2-insert motif of MCM3. We show that this has led to a competition between MCM3 and Nrf2 proteins for Keap1 binding, and likely recruited MCM3 for the competitive binding dependent modulation of Keap1 controlled Nrf2 activities. We hypothesise that such mechanism could help to adjust the Keap1-Nrf2 antioxidant response pathway according to the proliferative and replicative status of the cell, with possible reciprocal implications also for the regulation of cellular functions of MCM3. Altogether this suggests about important role of Keap1-MCM3 interaction in the cross-talk between replisome and redox homeostasis machineries in metazoan cells.

Two separate replication modes of the bovine papillomavirus BPV1 origin of replication that have different sensitivity to p53.

We have shown previously that transient amplificational replication of reporter plasmids that carry the papillomavirus origin of replication is efficiently blocked by p53 protein in several cell lines. We demonstrate now that the replication of stably maintained episomal bovine papillomavirus BPV1 URR (upstream regulatory region) reporter plasmid is not sensitive to p53. In addition, these two replication modes--initial transient amplificational replication and stable maintenance replication of essentially the same BPV1 URR reporter plasmid--can take place in the same cells, where amplificational replication does not interfere with the stable maintenance replication. These data suggest that BPV1 replicons could follow two clearly separable replication mechanisms during initial amplification and during stable extrachromosomal maintenance.

DNA binding polarity, dimerization, and ATPase ring remodeling in the CMG helicase of the eukaryotic replisome.

The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3' single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational and assembly states as it matures into a functional helicase.

The structural basis for MCM2-7 helicase activation by GINS and Cdc45.

Two central steps for initiating eukaryotic DNA replication involve loading of the Mcm2-7 helicase onto double-stranded DNA and its activation by GINS-Cdc45. To better understand these events, we determined the structures of Mcm2-7 and the CMG complex by using single-particle electron microscopy. Mcm2-7 adopts two conformations--a lock-washer-shaped spiral state and a planar, gapped-ring form--in which Mcm2 and Mcm5 flank a breach in the helicase perimeter. GINS and Cdc45 bridge this gap, forming a topologically closed assembly with a large interior channel; nucleotide binding further seals off the discontinuity between Mcm2 and Mcm5, partitioning the channel into two smaller pores. Together, our data help explain how GINS and Cdc45 activate Mcm2-7, indicate that Mcm2-7 loading may be assisted by a natural predisposition of the hexamer to form open rings, and suggest a mechanism by which the CMG complex assists DNA strand separation.

Brd4 is involved in multiple processes of the bovine papillomavirus type 1 life cycle.

Brd4 protein has been proposed to act as a cellular receptor for the bovine papillomavirus type 1 (BPV1) E2 protein in the E2-mediated chromosome attachment and mitotic segregation of viral genomes. Here, we provide data that show the involvement of Brd4 in multiple early functions of the BPV1 life cycle, suggest a Brd4-dependent mechanism for E2-dependent transcription activation, and indicate the role of Brd4 in papillomavirus and polyomavirus replication as well as cell-specific utilization of Brd4-linked features in BPV1 DNA replication. Our data also show the potential therapeutic value of the disruption of the E2-Brd4 interaction for the development of antiviral drugs.

Analysis of chromatin attachment and partitioning functions of bovine papillomavirus type 1 E2 protein.

Recent studies have suggested that the tethering of viral genomes to host cell chromosomes could provide one of the ways to achieve their nuclear retention and partitioning during extrachromosomal maintenance in dividing cells. The data we present here provide firm evidence that the partitioning of the bovine papillomavirus type 1 (BPV1) genome is dependent on the chromatin attachment process mediated by viral E2 protein and its multiple binding sites. On the other hand, the attachment of E2 and the E2-mediated tethering of reporter plasmids to host chromosomes are not necessarily sufficient for efficient partitioning, suggesting that additional E2-dependent activities might be involved in the latter process. The activity of E2 protein in chromatin attachment and partitioning is more sensitive to the point mutations in the N-terminal domain than its transactivation and replication initiation functions. Therefore, at least part of the interactions of the E2 N-terminal domain with its targets during the chromatin attachment and partitioning processes are likely to involve specific receptors not involved in transactivation and replication activities of the protein. The mutational analysis also indicates that the binding of E2 to chromatin is not achieved through interaction of linear N-terminal subsequences of the E2 protein with putative receptors. Instead, the composite surface elements of the N-terminal domain build up the receptor-binding surface of E2. In this regard, the interaction of BPV1 E2 with its chromosomal targets clearly differs from the interactions of LANA1 protein from Kaposi's sarcoma-associated human herpesvirus and EBNA1 from Epstein-Barr virus with their specific receptors.